The invention relates to new monoclonal antibodies specific for PHF-tau, to the hybridomas secreting these monoclonal antibodies, and to the antigen recognition pattern of these monoclonal antibodies. The invention also relates to a process for diagnosing brain diseases involving monoclonal antibodies of the invention, more particularly in cerebrospinal fluid (CSF) samples. The invention also relates to a region of the tau molecule modifiable in vivo by the process of phosphorylation, which is found to be associated with Alzheimer""s disease or with other types of dementia and which is specifically recognized by the monoclonal antibodies of the invention.
Alzheimer""s disease (AD) is the most common form of adult-onset dementia. At present, no reliable biochemical test is available for antemortem diagnosis of AD. The disease is therefore diagnosed clinically on the basis of exclusion of other forms of dementia. The diagnosis can be confirmed neuropathologically by the demonstration of large amounts of neuritic (senile) plaques and neurofibrillary tangles (NFT) in particular brain regions (McKhann et al, 1984).
Neurofibrillary tangles consist of paired helical filaments (PHFs). Immunocytochemical evidence suggests that the microtubule-associated protein tau is a major protein component of PHF and NFT (Brion et al., 1985b; Delacourte and Defossez, 1986; Grundke-Iqbal et al., 1986; Kosik et al., 1986; Wood et al., 1986). Definite proof that the tubulin-binding domain of tau is tightly associated with the core of PHFs was obtained via amino acid sequencing (Kondo et al., 1988). Nevertheless it has been suggested that tau peptides may represent only a small portion of the major component of PHF (Wischik et al., 1988).
Tau protein exists in different isoforms, of which 4 to 6 are found in adult brain but only 1 isoform is detected in fetal brain. The diversity of the isoforms is generated from a single gene on human chromosome 17 by alternative mRNA splicing (Andreadis et al., 1992). The most striking feature of tau protein, as deduced from molecular cloning, is a stretch of 31 or 32 amino acids, occurring in the carboxy-terminal part of the molecule, which can be repeated either 3 or 4 times. Additional diversity is generated through 29 or 58 amino acid-long insertions in the NH2-terminal part of tau molecules (Goedert et al., 1989). For simplicity, all numbering in this patent application refers to the tau variant htau40 containing all exons (441 amino acids long) according to Goedert et al (1989).
Under normal circumstances tau promotes microtubule assembly and stability in the axonal compartment of neurons. The microtubule-binding domain in tau is localized in the repeat region of tau (255-381) (Lewis et al, 1990) and is modulated by adjacent regions: the carboxyterminal tail (382-414) and the proline-rich region (143-254) (Drubin and Kirschner, 1991). Stability and bundling of the microtubules is mediated by a short hydrophobic zipper in the carboxyterminal tail of tau (Lewis et al, 1989). Both assembly and stability are regulated by alternative mRNA splicing and phosphorylation.
In normal circumstances adult brain contains 2 to 3 mol phosphate per mole of tau (Selden and Pollard, 1983; Ksiezak-Reding et al, 1992) present amongst others at serine 404 (Poulter et al, 1993), while other results demonstrate that phosphorylation of different sites in normal tau follows different developmental profiles (Lee et al, 1991; Bramblett et al, 1993; Goedert et al, 1993a). Abnormal tau variants of 60, 64 and 68 kDa have been detected exclusively in brain areas showing neurofibrillary changes and senile plaques (Delacourte et al. 1990). The abnormal electrophoretical behavior of tau is due to phosphorylation since alkaline phosphatase treatment of these tau molecules changes their molecular mass to that of normal tau (Goedert et al., 1992; Flament et al., 1990b, Greenberg and Davies, 1990). Currently abnormal phosphorylation sites have been detected in PHF-tau at positions 46, 231, 235, 263 and 396 (Iqbal et al., 1989; Lee et al., 1991; Hasegawa et al., 1992). In four of these sites, the phosphorylated residu is followed by a proline residu, indicating that a proline-directed kinase is involved in some of the abnormal phosphorylations of tau. In addition to these sites ten others are present in htau40, two of which are also abnormally phosphorylated, as indicated by antibody reactivity (Mab tau2: Watanabe et al., 1992; Mab AT8: Biernat et al., 1992, Goedert et al., 1993).
The abnormal phosphorylation of tau in Alzheimer""s disease is due to a shift in the phosphatase/kinase equilibrium. In vitro several kinases can phosphorylate tau: cdc2-kinases (Vulliet et al, 1992; Ledesma et al, 1992), MAP kinases (Drewes et al, 1992, Roder and Ingram, 1991), glycogen synthase kinases (Mandelkow et al, 1992) and TPKI and TPKII (Ishiguro et al, 1992). The phosphatases are less well studied in Alzheimer""s disease and so far only one phosphatase was able in vitro to dephosphorylate the abnormally phosphorylated sites, namely protein phosphatase 2A1 (Goedert et al, 1992).
So far, the detection of PHF-tau in brain extracts, either via antibodies (Mab Alz50: Ghanbari et al., 1990; Mab Ab423: Harrington et al., 1991), or via the change in molecular weight (Flament et al., 1990, Delacourte et al., 1993), or else by functional assay (Bramblett et al. 1992) has been very useful to discriminate dementia with altered cytoskeletal properties from normal aged subjects or from patients with other types of dementia. Nevertheless the detection of PHF-tau in CSF remained impossible, even using antibodies directed at one of the abnormally phosphorylated sites such as serine 202 (Goedert et al., 1993). This can be ascribed to one or more of the following reasons: 1) the low concentration of PHF-tau in CSF, 2) non-even use of phosphorylation sites among all the potential phosphorylation sites, 3) differences in phosphatase sensitivity of these sites, and, 4) too low an affinity constant of the antibodies used.
The aim of the present invention is therefore to provide monoclonal antibodies which allow the reliable and sensitive detection of abnormally phosphorylated tau present in cerebrospinal fluid.
The invention also aims at providing the hybridomas which secrete the above-said monoclonal antibodies.
The invention furthermore aims at providing the epitopes of the abnormally phosphorylated tau protein present in brain homogenates or in body fluids such as cerebrospinal fluid, which are recognized by said monoclonal antibodies.
Finally, the invention aims at providing a process for the detection or diagnosis in vitro of brain diseases involving abnormally phosphorylated tau proteins.
The present invention relates more particularly to a monoclonal antibody which forms an immunological complex with a phosphorylated epitope of an antigen belonging to abnormally phosphorylated tau (PHF-tau) residing in the region spanning positions 143-254 with the following amino acid sequence:
and with said monoclonal antibody being characterized by the the fact that it is capable of specifically detecting abnormally phosphorylated tau protein (PHF-tau) in cerebrospinal fluid (CSF).
The monoclonal antibodies of the invention were selected from a range of monoclonal antibodies obtained by direct immunization with PHF-tau, extracted from human brain tissue derived from Alzheimer patients. More particularly the monoclonal antibodies of the invention are characterized by the fact that they specifically bind to naturally occuring abnormally phosphorylated tau. Further analysis of their epitopes showed that the monoclonal antibodies of the invention are directed at phosphorylated epitopes confined to a particular region of the tau molecule, namely the region between 143 and 254 including several potential phosphorylation sites such as T153 and S235 used by the SP and TP directed kinases. The monoclonal antibodies of the invention are further characterized by the fact that they recognize epitopes which are different from the epitope of the monoclonal antibody AT8 as defined in Goedert et al. (1993) and upon comparison with the AT8 antibodies allow the detection of PHF-tau in CSF. They recognize preferentially PHF-tau either on brain sections, immunoblots or in ELISA and they are surprisingly able to detect PHF-tau in CSF, either alone or in combination with other PHF-tau specific antibodies.
In conclusion, the monoclonal antibodies of the invention are characterized in that they specifically bind to naturally occuring abnormally phosphorylated tau of which the phosphorylation state is confined to a particular region of the tau molecules as specified above, or bind to recombinant non-phosphorylated tau after treatment with proline-directed kinases, which can provoke the phosphorylation of, amongst others, Ser-Pro or Thr-Pro sites in the region as specified. Proline-directed kinases such as MAP kinases (Sturgill et al., 1991). cdc2 kinases (Labbxc3xa9 et al., 1991) and glycogen synthase kinases (Vandenheede et al., 1980) can be purified from various tissues or can be present in brain extracts. The phosphorylation of tau by these kinases is abolished or greatly diminished when one or more of the following serines/threonines are mutated to an amino acid such as Ala: T153, T175, T181, S199, S202, T205, T212, T217, T231, or S235. Consequently, the epitope of these antibodies can be characterized via such mutant tau, or via non-phosphorylated tau such as procaryotically expressed recombinant tau and their phosphorylated homologues, or via synthetic peptides having the same amino acid sequence as parts of the region specified above of the human tau 40 protein and with said peptides being capable of being phosphorylated by said kinases or being incapable of being phosphorylated upon synthesis of the peptides. The epitopes of the present invention are thus defined as the proline rich-region of tau between position 143 and 254 and which can be abnormally phosphorylated at threonine 153 (T153), T175, T181, S199, S202, T205, T212, T217, T231 and S235 or a combination these sites included in the epitope of these antibodies, further referred to as a xe2x80x9cPHF-tau epitopesxe2x80x9d.
The expression xe2x80x9cspecifically detecting abnormally phosphorylated tau proteinxe2x80x9d corresponds to the fact that the monoclonal antibodies of the invention detect abnormally phosphorylated tau in CSF without cross-reacting with normal tau present in CSF.
The expression xe2x80x9cform an immunologically complex withxe2x80x9d means that the monoclonal antibody of the invention binds to the above-said antigen under conditions as mentioned in one of the following techniques:
Light Immunomicroscopy
Brain tissue samples, of e.g. Alzheimer patients obtained at surgery or autopsy, are fixed by immersion in 4% formalin or Bouin""s fixative and embedded in paraffin for sectioning. The monoclonal antibodies of the invention are applied in conjunction with a technique to visualize the formed immune complexes such as the avidin-biotinylated peroxidase complex technique (Hsu et al., 1981) using 3,3xe2x80x2-diaminobenzidine tetrahydrochloride for development of color. Sections are counterstained with Harris haematoxylin stain.
Immunoelectron Microscopy in Tissue Sections
Brain tissue samples e.g. obtained from Alzheimer patients at surgery or autopsy are fixed in either Bouin""s fixative or 10% buffered formalin before sectioning without embedding (Vibratome). The monoclonal antibody of the invention is used for immunostaining by the indirect immunogold method after which the sections are fixed, embedded and sectioned for electron microscopy, all according to standard protocols known to those skilled in the art (Brion et al., 1985a).
Immunoblotting Procedures
For immunoblotting, fractions enriched in PHF-tau are prepared as described (Greenberg and Davies, 1990). Typically, postmortem tissue, consisting mostly of gray matter from the frontal and temporal cortex, was obtained from histologically confirmed Alzheimer patients. This Alzheimer gray matter brain sample (5-10 g) was homogenized with 10 volumes of cold buffer H (10 mM Tris/1 mM EGTA/0.8 M NaCl/10% sucrose, pH 7.4) in a Teflon/glass Potter S (Braun, Germany) homogenizer. After centrifugation of the homogenate in a 60 Ti MSE rotor at 27,000xc3x97g for 20 min at 4xc2x0 C., the pellet was removed and the supernatant was adjusted to 1% (wt/vol) N-laurosylsarcosine and 1% (vol/vol) 2-mercaptoethanol and incubated while rotating on a mixer (Swelab, Sweden) for 2.5 hours at 37xc2x0 C. The supernatant mixture was centrifuged at 108,000xc3x97g for 35 min at 20xc2x0 C. The PHF-tau containing pellet was gently washed with PBS and finally suspended in 1 ml of the same buffer.
SDS-polyacrylamide electrophoresis is performed under reducing conditions on 12% gels (Laemmli, 1970). After electrophoresis, the proteins are either fixed and stained with Coomassie brilliant blue, or transferred (Towbin et al., 1979) to nitrocellulose sheets (Hybond-C, Amersham) or Immobilon filters (Millipore).
After transfer, the filters are presoaked in PBS containing 0.05% (v/v) Tween 20 (Tween-PBS) and then incubated for 1 h in Tween-PBS containing 5% (w/v) skimmed dried milk and 10% (v/v) newborn calf serum (blocking buffer). Next, the filters are treated overnight at 4xc2x0 C. with a monoclonal antibody according to the invention appropriately diluted in blocking buffer.
The filters are then washed three times in Tween-PBS and treated for 1.5 h at room temperature with horseradish peroxidase-labeled rabbit anti-mouse IgG (Dakopatts, Denmark) diluted 1/3000 in blocking buffer. After three washes in Tween-PBS, streptavidine-biotinylated horseradish peroxidase complex (Amersham), diluted 1/250 in blocking buffer, is applied for 1.5 h at room temperature. Thereafter, the filters are washed three times in Tween-PBS and once in PBS. The filters are then incubated in PBS containing 0.05% (w/v) diaminobenzidine and 0.03% (v/v) hydrogen peroxide until background staining develops.
It should be clear that the formation of an immunological complex between the monoclonal antibodies and the antigen is not limited to the precise conditions described above, but that all techniques that respect the immunochemical properties of the antibody and antigen binding will produce similar formation of an immunological complex.
The present invention relates more particularly to a monoclonal antibody as defined above, characterized by the fact it forms an immunological complex:
either with a phosphorylated epitope located within the sequence defined above (SEQ ID NO 1),
or with any other phosphorylated peptide capable of forming an immunological complex with a monoclonal antibody, which itself is capable of forming a complex with a phosphorylated epitope located in the human tau protein region as shown in SEQ ID NO 1.
Preferred monoclonal antibodies of the invention, AT180 and AT270, are produced by hybridomas deposited at ECACC (European Collection of Animal Cell Cultures, Vaccine Research and Production Laboratory, Public Health and Laboratory Service (PHLS), Centre for Applied Microbiology and Research, Porton Down, GB-Salisbury, Wiltshire SP4 OJG), United Kingdom on Dec. 22, 1992, under No 92122204 or on Jul. 7, 1993 under No 93070774.
The above-mentioned monoclonal antibodies are obtained by a process involving obtention and isolation of hybridomas which secrete these monoclonal antibodies.
The preferred monoclonal antibodies of the invention allow the detection of at least 1, 5, 10 or 20 pg/ml phosphorylated tau as determined in an ELISA using these monoclonal antibodies in the coating phase and incubating them with CSF spiked with different amounts of phosphorylated and non-phosphorylated tau without amplification. Phosphorylated tau is prepared by incubating recombinant non-phosphorylated tau with a rat brain extract capable of phosphorylation of Ser and Thr amino acids at positions corresponding to sites of abnormal phosphorylation of tau (Goedert et al., 1993), as found in tau extracts of brain tissue derived of patients having died of Alzheimer""s disease.
A process for obtaining the hybridomas of the invention involves:
starting from spleen cells of an animal, e.g. mouse or rat, previously immunized in vivo or from spleen cells of such animals previously immunized in vitro with an antigen being preferably abnormally phosphorylated tau (PHF-tau), or a phosphorylated human tau peptide or immunoaffinity purified abnormally phosphorylated tau, as disclosed below, recognized by the monoclonal antibodies of the invention;
fusing said immunized cells with myeloma cells under hybridoma-forming conditions; and
selecting those hybridomas which secrete the monoclonal antibodies which are capable of specifically recognizing a phosphorylated epitope of abnormally phosphorylated tau (PHF-tau) in cerebrospinal fluid (CSF).
The phosphorylated human tau peptide refers to a peptide comprising in its amino acid sequence a phosphorylated sequence comprised in the region spanning amino acids 143 to 254 of human tau and with said peptide being characterized by the fact that it can form an immunological complex with the antibodies of the invention.
The antigen of the invention is advantageously contained in the brain, in the cerebrospinal fluid or the serum of a patient having Alzheimer""s disease, Down""s syndrome, Pick""s disease, subacute sclerosing panencephalitis (SSPE) or other neurological diseases in which the abnormally phosphorylated tau protein is implicated; this antigen provokes an immunological reaction with the monoclonal antibody of the invention.
More particularly, the present invention relates also to monoclonal antibodies as defined above, obtained by a process such as as defined above, characterized in that it involves:
starting from the spleen cells of a mouse previously immunized with abnormally phosphorylated tau (PHF-tau) extracted and purified from a human brain sample of a patient suffering from Alzheimer""s disease (as disclosed in the examples section), or a phosphorylated human tau peptide, or immunoaffinity-purified abnormally phosphorylated tau capable of reacting with the monoclonal antibodies of the invention,
fusing said immunized cells with myeloma cells under hybridoma-forming conditions,
selecting those hybridomas which secrete monoclonal antibodies which specifically recognize PHF-tau and which are capable of specifically detecting PHF-tau in CSF (as illustrated in detail in the examples section).
A process for producing the monoclonal antibodies of the invention involves:
culturing the selected hybridomas as indicated above in an appropriate culture medium; and
recovering the monoclonal antibodies secreted by said selected hybridoma; or alternatively
implanting the selected hybridoma into the peritoneum of a mouse and, when ascites has been produced in the animal;
recovering the monoclonal antibodies then formed from said ascites.
The monoclonal antibodies of the invention can be prepared by conventional in vitro techniques such as the culturing of immobilized cells using e.g. hollow fibers or microcapsules or such as the culturing of cells in homogeneous suspension using e.g. airlift reactors or stirred bioreactors.
The invention also relates to a peptide capable of forming an immunological complex with any of the monoclonal antibodies of the invention, with said peptide being in the phosphorylated form, and,
with the sequence of said peptide comprising, or consisting of phosphorylated parts of the sequence as shown in SEQ ID NO 1, or,
with the sequence of said peptide comprising, or consisting of the sequence of any peptide being capable of forming an immunological complex with anyone of the monoclonal antibodies according to the invention.
Said phosphorylated peptides are preferably from 6 to 100 amino acids long. The peptides according to this embodiment of the invention can be prepared by classical chemical synthesis. The synthesis may be carried out in homogenous solution or in solid phase according to any of the techniques well known in the art.
Phosphorylated peptides are prepared according to any technique known in the art, (f.i. de Bont et al., 1990a; de Bont et al., 1990b; Perich, 1991; Orvos et al., 1989).
According to a preferred embodiment, the present invention relates to a phospohorylated peptide as defined above consisting of or comprising in its amino acid sequence the following sequence:
Val-Arg-Thr-Pro-Pro (amino acid 229-233; human tau 40 numbering, SEQ ID NO 2), with said Thr(231) being phosphorylated and with said peptide being characterized in that it is able to form an immunological complex with the monoclonal antibody AT180 produced by the hybridoma deposited at the ECACC on Dec. 22, 1992 under No. 92122204.
According to another preferred embodiment, the present invention relates also to a phosphorylated peptide as defined above consisting of or comprising in its amino acid sequence the following sequence:
Pro-Lys-Thr-Pro-Pro (amino acid 179-183; human tau 40 numbering; SEQ ID NO 3), with said Thr(181) being phosphorylated and with said peptide being characterized in that it is able to form an immunological complex with the monoclonal antibody AT270 produced by the hybridoma deposited at the ECACC on Jul. 7, 1993 under No. 93070774.
According to yet another embodiment, the present invention relates to a phosphorylated peptide as defined above, which is capable of generating a monoclonal antibody according to any one of claims 1 to 4 upon immunization.
The peptides used for immunization are preferentially in the form in which they are joined to a carrier molecule in order to achieve a good immunogenic response. Such carrier molecules are well known in the art and are coupled to the peptide via linker groups, which are also comprised in the art.
The invention also relates to a process for the post-mortem detection or diagnosis in vitro of a brain/neurological disease involving PHF-tau, such as Alzheimer""s disease, which comprises at least the following steps:
contacting a monoclonal antibody of the invention with a preparation of NFT or a detergent-extracted brain homogenate isolated from a patient having had Alzheimer""s disease or any other disease involving abnormally phosphorylated tau protein (PHF-tau) under conditions suitable for producing an antigen-antibody complex;
detecting the immunological binding of said antibody to said brain homogenate, and possibly separating said complex and recovering the antigen sought in a purified form.
Recovering the antigen sought may be done by first washing the immobilized antibody-antigen complex then formed;
treating this complex with a solution (e.g. 3 M potassium thiocyanate, 2.5 M magnesium chloride, 0.2 M citrate-citric acid, pH 3.5 or 0.1 M acetic acid) capable of producing the dissociation of the antigen-antibody complex; and;
recovering the antigen in a purified form.
The invention relates also to a process for the detection or diagnosis in vitro of brain/neurological disease involving abnormally phosphorylated tau protein, such as in Alzheimer""s disease, which includes:
bringing a sample of CSF, more preferably unconcentrated CSF, or a sample of serum from a patient suspected of suffering from brain disease involving PHF-tau, more particularly Alzheimer""s disease, or proteins or polypeptides as a result of an extraction procedure starting from brain tissues, cerebrospinal fluid or serum known to those skilled in the art (Ibqal et al., 1984; Greenberg and Davies, 1990) into contact under in vitro conditions with a monoclonal antibody of the invention, with said conditions being suitable for producing an antigen-antibody complex; and,
detecting the immunological binding of said antibody to said sample of brain extract, cerebrospinal fluid or serum, or proteins or polypeptides.
Advantageously, the monoclonal antibodies of the invention are in an immobilized state on a suitable support such as a resin. Alternatively, the present process may be put into practice by using any other immunoassay format known to the person skilled in the art.
The process for the detection of the antigen can then be carried out for instance as follows:
bringing together said antigen-antibody complex formed by the antigen and the antibodies of the invention with:
a second antibody
which can be a monoclonal antibody recognizing an epitope of abnormally phosphorylated tau protein, or an epitope of any phosphorylated tau peptide carrying an epitope, with said epitopes being different from the one of the invention, or
which can be a polyclonal antibody recognizing abnormally phosphorylated tau or a polyclonal antibody recognizing a peptide carrying an epitope of PHF-tau, with said polyclonal antibody being capable of forming an immunological complex with epitopes which are different from the epitope of the invention, with said polyclonal antibody being preferably purified by immunoaffinity chromatography using immobilized tau protein;
a marker either for specific tagging or coupling with said second antibody, with said marker being any possible marker known to the person skilled in the art;
appropriate buffer solutions for carrying out the immunological reaction between the monoclonal antibody of the invention and a test sample on the one hand, and the bound second antibody and the marker on the other hand.
The detection of the immunologically bound monoclonal antibody can be achieved by conventional technology comprised in the art. Advantageously, the second antibody itself carries a marker or a group for direct or indirect coupling with a marker.
The monoclonal antibodies of the invention also enable the diagnosis of Alzheimer""s disease (AD) and of any disease involving the formation of abnormally phosphorylated tau in the region 143 to 254 on the basis of CSF (i.e. to detect modified forms of tau in CSF). The problem associated herewith is that this antigen is present in a very low amount in CSF, so the detection assay must be very sensitive. This sensitivity problem may be further overcome by (i) using a combination of the monoclonal antibodies of the invention, or (ii) a combination of a monoclonal antibody of the invention with any other normal and/or abnormally phosphorylated tau monoclonal antibodies known in the art and/or (iii) by using a monoclonal antibody or a combination of monoclonal antibodies of the invention in combination with an amplification technique such as the catalyzed reporter deposition amplification technique (CARD, Bobrow et al., 1989), allowing a PHF-tau specific ELISA with a higher sensitivity.
The results obtained with the monoclonal antibodies of the invention indicate that elevated PHF-tau levels are found in AD, but may occur also in other neurological diseases where abnormal phosphorylation of tau occurs in the region of tau comprised by amino acids 143 to 254.
According to another embodiment, the present invention relates to a kit for the diagnosis in vitro of one of the following diseases: Alzheimer""s disease, Down""s syndrome, Pick""s disease and other neurological disorders in which abnormally phosphorylated tau protein or paired helical filaments are implicated, characterized in that the kit comprises:
at least one monoclonal antibody of the invention deposited on a microplate;
a preparation containing the sample (CSF, serum or the proteins extracted therefrom) to be diagnosed in vitro,
a second antibody
which can be a monoclonal antibody recognizing an epitope of abnormally phosphorylated tau protein, or an epitope of any phosphorylated tau peptide carrying an epitope, with said epitopes being different from the one of the invention, or
which can be a polyclonal antibody recognizing abnormally phosphorylated tau or a polyclonal antibody recognizing a peptide carrying an epitope of PHF-tau, with said polyclonal antibody being capable of forming an immunological complex with epitopes which are different from the epitope of the invention, with said polyclonal antibody being preferably purified by immunoaffinity chromatography using immobilized tau protein;
a marker either for specific tagging or coupling with said second antibody;
appropriate buffer solutions for carrying out the immunological reaction between the monoclonal antibody of the invention and a test sample on the one hand, and the bound second antibody and the marker on the other hand,
possibly a peptide carrying an epitope of PHF-tau comprised in the region spanning amino acids 143 to 254 for standard purposes, or for competition purposes with respect to the antigen which is sought.
A preferred embodiment of the present invention for the detection or diagnosis in vitro of brain/neurological disease involving abnormally phosphorylated tau protein, such as Alzheimer""s disease relates to a method or a kit as defined above, which comprises a mixture (combination) of monoclonal antibodies of the invention, or a combination of at least one monoclonal antibody of the invention with other antibodies capable of specifically recognizing a region of PHF-tau residing in the region spanning positions 143-254 of human tau 40 (SEQ ID NO 1), with said monoclonal antibodies being preferably chosen from:
(1) the monoclonal antibody AT180 produced by the hybridoma deposited at the ECACC on Dec. 22, 1992 under No. 92122204;
(2) the monoclonal antibody AT270 produced by the hybridoma deposited at the ECACC on Jul. 7, 1993 under No. 93070774;
(3) the monoclonal antibody AT8 produced by the hybridoma deposited at the ECACC on Oct. 8, 1991 under No. 91100806;
and with said mixture being preferably chosen from the following list:
a mixture of monoclonal antibodies comprising the monoclonal antibodies (1) and (2),
a mixture of the monoclonal antibodies comprising the monoclonal antibodies (1) and (3),
a mixture of the monoclonal antibodies comprising the monoclonal antibodies (2) and (3),
a mixture of the monoclonal antibodies comprising the monoclonal antibodies (1), (2) and (3);
with said method or kit being further characterized as containing or using:
a preparation containing the sample to be diagnosed in vitro;
a second antibody
which can be a monoclonal antibody recognizing an epitope of abnormally phosphorylated tau protein, or an epitope of any phosphorylated tau peptide carrying an epitope, with said epitopes being different from the one of the invention, or
which can be a polyclonal antibody recognizing abnormally phosphorylated tau or a polyclonal antibody recognizing a peptide carrying an epitope of PHF-tau, with said polyclonal antibody being capable of forming an immunological complex with epitopes which are different from the epitope of the invention, with said polyclonal antibody being preferably purified by immunoaffinity chromatography using immobilized tau protein;
a marker either for specific tagging or coupling with said second antibody;
appropriate buffer solutions for carrying out the immunological reaction between the monoclonal antibody of the invention and a test sample on the one hand, and the bound second antibody and the marker on the other hand,
possibly a peptide carrying an epitope of PHF-tau for standard purposes, or for competition purposes with respect to the antigen which is sought.
According to yet another preferred embodiment, the present invention relates to a method or kit for detecting or diagnosing in vitro a brain/neurological disease involving abnormally phosphorylated tau protein, such as Alzheimer""s disease, which involves a sandwich ELISA detection format comprising coating and detecting antibodies, with said coating antibodies consisting of at least one monoclonal antibody of the invention, and with said detecting antibodies consisting of at least one monoclonal antibody capable of detecting normal and/or abnormally phosphorylated human tau of which the epitope is different from any of the epitopes of the monoclonal antibodies of the invention. Such a preferred sandwich ELISA format is extensively illustrated in the examples section of the present invention.
Table 1
Detection of PHF-tau and normal tau using PHF-tau specific monoclonal antibodies AT180 and AT270. Microplates coated with saturating amounts of a monoclonal antibody specifically recognizing PHF-tau were incubated with CSF spiked with different amounts of non-phosphorylated or phosphorylated tau, the latter prepared by incubating recombinant non-phosphorylated tau with a rat brain extract capable of phosphorylating Ser and Thr amino acids at positions corresponding to the sites of abnormal phosphorylation of tau (Goedert et al., 1993). Bound antigen was detected as described in the examples section.
Table 2
CSF samples from AD patients, control patients and patients suffering from various non-AD neurological disorders were tested in ELISA using different combinations of capturing antibodies as described (example III). All values are expressed as mOD units except for the determination of total tau which was done using the Innotest htau (Innogenetics, Belgium) and which are expressed in pg/ml CSF.
The different experimental conditions used for each set-up do allow only intra-lane comparison.
Table 3
CSF samples of control patients, AD patients and patients suffering from various non-AD neurological disorders (OND) were assayed using the Innotest htau (Innogenetics, Belgium). From the cohorts of AD patients and OND patients those having high total tau values were selected for further testing using the PHF-tau specific ELISA in which AT8, AT180 and AT270 were used as capturing antibodies and AT120 and HT7 as detecting antibodies as described (Example IV). Results are expressed in pg/ml tau in CSF for total tau (Innotest htau) and as mOD units for the PHF-tau specific ELISA.
Figure 1
Detection of PHF-tau and normal tau using monoclonal antibody AT180. Microplates coated with saturating amounts of monoclonal antibody AT180 specifically recognizing PHF-tau were incubated with CSF spiked with different amounts of non-phosphorylated or phosphorylated tau, the latter prepared by incubating recombinant non-phosphorylated tau with a rat brain extract capable of phosphorylating Ser and Thr amino acids at positions corresponding to the sites of abnormal phosphorylation of tau (Goedert et al., 1993). Bound antigen was detected as described in the examples section.
Figure 2
Detection of PHF-tau and normal tau using monoclonal antibody AT270. Microplates coated with saturating amounts of monoclonal antibody AT270 specifically recognizing PHF-tau were incubated with CSF spiked with different amounts of non-phosphorylated or phosphorylated tau, the latter prepared by incubating recombinant non-phosphorylated tau with a rat brain extract capable of phosphorylating Ser and Thr amino acids at positions corresponding to the sites of abnormal phosphorylation of tau (Goedert et al., 1993). Bound antigen was detected as described in the examples section.
Figure 3
Phosphorylation of wild-type and mutated recombinant tau (expressed from clone human tau 24; Goedert and Jakes, 1990) with the protein kinase activity from rat brain. Immunoblots with anti-tau antiserum 134 and monoclonal antibodies AT8 and AT180. Lanes 1. tau 24; 2, tau 24 plus brain extract; 3, T231 A tau 24; 4, T231 A tau 24 plus brain extract, 5, S235 A tau 24; 6, S235 A tau 24 plus brain extract.
Figure 4
Phosphorylation of wild-type and mutated recombinant tau (expressed from clone human tau 24) with the protein kinase activity from rat brain. Immunoblots with anti-tau antiserum 134 and monoclonal antibodies AT8 and AT270. Lanes 1, tau 24; 2, tau 24 plus brain extract; 3, T175 A tau 24; 4, T175 A tau 24 plus brain extract; 5, T181 A tau 24; 6, T181A tau 24 plus brain extract.